Polarized transporting epithelia comprise tightly adherent cell monolayers that form selective permeability barriers between different biological compartments in the body and regulate ionic homeostasis by vectorial transport of ions and solutes between those compartments. Structural and functional abnormalities of epithelia are characteristic of many human diseases. The long-term goals of our work are to understand how epithelial cells organize into monolayers through specialized cadherin-mediated cell-cell contacts, and localize proteins to functionally different plasma membrane domains. We have integrated different experimental approaches to address these problems: structural analysis of proteins and protein complexes, high-resolution live cell imaging, biochemical analysis of protein complex assembly and function in cells, and in vitro reconstitution of protein functions. During the previous funding period, we defined stages in cell-cell adhesion and obtained preliminary evidence of a mechanism involved, determined how several plasma membrane proteins are targeted to and organized in specific membrane domains, and developed new in vitro methods to dissect protein interactions and functions at cell-cell contact plasma membranes. We translated these results and conclusions to studies of ischemic damage in kidney transplant patients. Our working hypothesis is that cell-cell adhesion establishes spatial cues for localized assembly of protein complexes to a Junctional Signaling Nexus (Par complex) that controls cell-cell adhesion (cadherin/catenins), delivery of post-Golgi transport vesicles (Sec6/8 complex), mitotic spindle orientation, and tight junction function. We will test this hypothesis by: 1). Define molecular mechanisms of cadherin-mediated cell-cell adhesion, 2). In vitro reconstitute cadherin/catenin-actin cytoskeleton interactions, 3). Mechanism of vesicle docking/fusion to cadherin-based plasma membrane domains, and 4). Analyze the Par complex, and assembly/function of a Junctional Signaling Nexus. The significance of these studies is that results will define molecular linkages and mechanisms controlling membrane domain formation by cadherinbased cell-cell adhesions, and provide a base line for understanding abnormalities in epithelial cell functions in different disease states.